Bibliographic details numerically referred to in this specification are collected at the end of the description.
The reference to any prior art in this specification is not and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.
Coagulation is an important mechanism in arresting bleeding and is a life-sustaining process. The two major arms of the blood coagulation cascade, the intrinsic and extrinsic pathways, consist of a series of stepwise, coordinated reactions involving specific plasma proteins in a process leading to thrombin generation which is in turn responsible for the conversion of fibrinogen to an impermeable cross-linked fibrin clot.
Blood coagulation or clotting takes place in three central phases. The first phase is the activation of a prothrombin activator complex. The second phase is the activation of prothrombin. The third stage is clot formation as a result of fibrinogen cleavage by activated thrombin.
The intrinsic and extrinsic pathways each lead to a different form of the prothrombin activator. The intrinsic mechanism of prothrombin activator formation begins with trauma to the blood or exposure of blood to collagen in a traumatised vessel wall. This usually also results in damage to fragile platelets. The cascade begins with the activation of factor XII (XIa) and the release of platelet factor 3 (PF3) from damaged platelets. Activated factor XII (requires prekallikrein and kininogen) cleaves and activates factor XI to become factor XIa. Activator factor XI converts factor IX to become activated factor IX (IXa) and factor IXa converts factor X to activated factor X (Xa). Calcium ions are required for the first three steps. Factor Xa then activates the common pathway of coagulation.
The extrinsic mechanism of prothrombin activator formation begins with trauma to vascular walls or extravascular tissues. The damaged tissue releases tissue thromboplastin also known as tissue factor (TF). The formation of a clot by this mechanism usually takes as little as 15 seconds. The cascade is initiated by the activation of factor X by TF and factor VII. Factor VIIa also activates factor IX in the presence of tissue factor, providing a connection between the “extrinsic” and “intrinsic” pathways. Factor Xa combined with factor V, factor VII and tissue factor constitutes the prothrombin activator. Calcium ions are required for each of these steps.
The common pathway of coagulation starts with the conversion of factor X to activated factor X described in the above paragraphs by the intrinsic and extrinsic pathways. Activated factor X requires its own cofactors for activity, including calcium ions, circulating factor V and an electrically charged platelet surface for localisation. It is then able to cleave prothrombin to produce activated thrombin. Thrombin converts fibrinogen (soluble) to fibrin (insoluble) and activates factor VIII. A network of insoluble fibrin (stabilised by thrombin) is formed, which is localised to the site of injury and traps oncoming blood platelets and plasma to form a clot.
The physiological function of coagulation is to prevent the loss of blood after injury and is part of a mechanism called haemostasis which is the result of a complex balance between the processes of fibrin clot initiation, formation and dissolution. However, certain events such as damage to the vessel wall or changes in blood flow can upset the balance and produce changes in the processes of coagulation that result in abnormal clot formation (thrombosis) in blood vessels.
Thrombosis is a pathological process in which a platelet aggregate and/or fibrin clot forms in the lumen of an intact blood vessel or in a chamber of the heart. If thrombosis occurs in an artery, myocardial infarction and unstable angina may result as a result of the tissue supplied by the artery undergoing ischaemic necrosis. Thrombosis formation in venous vasculature may result in a pulmonary embolism due to reduced blood flow. Disseminated intravascular coagulopathy in both the venous and arterial systems commonly occurs during septic shock, some viral infections and cancer which often leads to rapid and widespread thrombus formation and organ failure.
Current anticoagulant therapies such as heparin and warfarin, while effective, have several limitations such as an elevated risk of bleeding and inconvenience posed by the need for routine coagulation monitoring and/or parenteral administration. Heparin for example, is limited by the requirement for parenteral administration, constant monitoring, narrow therapeutic window, heparin rebound, thrombocytopaenia and bleeding. Warfarin, similarly, can lead to bleeding and may require constant monitoring due to its narrow therapeutic range and somewhat unpredictable effect. Thus, there is still a need to develop compounds or substances which have improved efficacy, safety and ease of use.
Human apolipoprotein CIII is a 8.8 kD protein glycosylated at Thr74 and synthesized in the liver and intestine. It is part of the apolipoprotein C family which also includes apolipoprotein CI and apolipoprotein CII. Apolipoprotein CIII plays a central role in modulating metabolism of triglyceride-rich plasma lipoproteins and levels in normal human plasma are 100-150 μg/ml. It is associated predominantly with triglyceride-rich very low density lipoprotein (VLDL). Some apolipoprotein CIII are associated with high density lipoprotein (HDL). In man, plasma triglyceride levels are positively associated with apolipoprotein CIII levels. Transgenic overexpression in mice results in hypertriglyceridemia (Ito Y., Science, 249: 790-793, 1990). Apolipoprotein CIII gene knockout mice are hypotriglyceridemic (Maeda N. et al., J. Biol. Chem., 269: 23610-23616, 1994). Apolipoprotein CIII inhibits lipoprotein lipase activity and reduces uptake and clearance of triglyceride-rich lipoproteins by the liver. Taken together, there is strong evidence that increased plasma levels of apolipoprotein CIII contribute to the development of hypertriglyceridemia in man (for review, see; Mahley, R. W. et al., J. Lipid Res., 25: 1277, 1984; Jong, M. C. et al., Arterioscler. Thromb. Vasc. Biol., 19: 472, 1999; Breslow, J. Proc. Natl. Acad. Sci., USA, 90:8314, 1993). Human apolipoprotein CIII exists in three forms depending upon the level of sialylation: C-III0. C-III1, and C-III2. The subscript indicates the number of sialic acid residues, however, the C-III0 form does not include the neutral carbohydrates. Glycosylation occurs on threonine (T) at position 74.
Several human apolipoprotein CIII polymorphisms have been described. Thrombin cleavage of apolipoprotein CIII into two fragments, 1-40 and 41-79 suggests that the C-terminal 41-79 peptide can bind phospholipid (Sparrow J. T. et al., Biochemistry 16:5427-31, 1977). Synthetic apolipoprotein CIII peptides suggest that the minimal sequence required for phospholipid binding is contained within amino acids 48-79 (Sparrow J. T. and Gotto A. M., CRC Crit. Rev. Biochem. 13: 87-107, 1982). Inhibition of lipoprotein lipase activity is mediated by the N-terminal domain of apolipoprotein CIII (McConathy W. J. et al., J. Lipid Res. 33: 995-1003, 1992).
In work leading up to the present invention, the inventors determined that a fragment of apolipoprotein CIII (SEQ ID NO: 2), being a polypeptide comprised of amino acids 41-79 (SEQ ID NO: 4) prolonged induction of blood coagulation in in vitro prothrombin time assays. Such results indicate that the fragment of apolipoprotein CIII is capable of inhibiting blood coagulation by inhibiting the extrinsic coagulation pathway.